DNA Origami Folding Pathways: Chemical Quenching and Competitive Anneals

Jacob Majikes
North Carolina State University

I present a new workbench scaffold (miniM13) and two new techniques to probe the folding and formation of DNA nanostructures which can be implemented with standard equipment available to DNA nanotechnology labs. The use of this smaller scaffold allows faster iteration through design/prototype cycles, and facilitates experiments which explore diverse nanostructure designs.
The first new technique addressed in this presentation is that of competitive multishape anneals, in which one scaffold is folded simultaneously by multiple staple sets. Competitive anneals allow examination of systemic folding preferences via which sections of a structure ‘win’ over their competition and are present in the final structure. Three shapes were brought into competition and were examined with AFM, image analysis, and fluorescence spectroscopy. Their results implicate the role of conformational entropy in the annealing process, and were used to inform preliminary routing patterns and predictive phase diagrams for these systems.
The second technique is to “chemically freeze”, or quench, DNA nanostructure formation at different times during the folding process. Given the difficulty and limitations of techniques to probe intermediate folding states, the chemical quench presents an elegant solution for arbitrary nanostructure folding pathways. Results of chemical quenching the standard Tall Rectangle nanostructure further implicate the dominance of conformational entropy in structure formation.